Reversed-phase ODS Column Research Articles

Comparison of Kinins Derived from Rat Stomach Tissue and Produced by Catheptic Enzyme in Rat Stomach

Four uterine-contractile substances (a, b, c and d) and three uterine-contractile substances (a′, c′ and d′) were obtained from the reaction mixture of rat plasma kininogen with the kinin-forming enzyme in rat stomach and the homogenate of the rat stomach with 0.2% acetic acid, respectively. By high performance liquid chromatography with a Zorbax ODS reversed-phase column, the retention times of materials a, c and d were found to be equal to those of materials a′, c′ and d′, and they were not equal to those of kallidin, methionyl-lysyl-bradykinin (MLBK) and bradykinin (BK). The retention time of material b was equal to that of BK. The uterine-contractile activities of the materials a, b, c, d, a′, c′ and d′ were all abolished by chymotrypsin, but not by trypsin treatment. These materials all relaxed the isolated rat duodenum in the presence of atropine, dibenamine, diphenhydramine and propranolol, and they produced a fall in rabbit blood pressure after administration of atropine, dibenamine and propranolol. The content ratios of a: b: c: d and a′: c′: d′ were 4: 4: 67: 25 and 65: 19: 16, respectively, as calculated from the uterine-contractile activities of these materials.

Separation of alkenylacyl, alkylacyl, and diacyl analogues and their molecular species by high performance liquid chromatography.

High performance liquid chromatography methods were established for separation of alkenylacyl, alkylacyl, and diacyl acetylglycerols derived from ethanolamine glycerophospholipids (EGP) and for separation of the individual molecular species from each of the separated classes. The EGP were isolated from bovine brain, hydrolyzed with phospholipase C, and acetylated with acetic anhydride. The three classes of diradylacetylglycerols were separated quantitatively on a mu Porasil silica column. Individual classes were further fractionated on a Zorbax ODS reverse phase column. By gas--liquid chromatographic quantitation of each peak, 29-33 different molecular species were identified within each class. For alkenylacyl-GPE, the major species were 18:1-18:1, 21.8%, and 16:0-18:1, 14.8%. Polyenoic fatty acids predominated at the 2-position of diacyl-GPE. The major species were 18:0-22:6 (n-3), 25.5%, and 18:0-20:4 (n-6), 15.8%. Three species of alkylacyl-GPE, 18:0-20:6 (n-3), 16:0-22:4 (n-6), and 18:0-22:4 (n-6), each accounted for 10%.

Simultaneous Determination of Folpet, Piperonyl Butoxide, and Pyrethrins in Aerosol Formulations by High Pressure Liquid Chromatography

Abstract A high pressure liquid chromatographic (HPLC) method is described for the simultaneous determination of folpet, piperonyl butoxide, and pyrethrins in aerosol pesticide formulations. The aerosol propellants are removed from the can and the remaining components are determined by HPLC, using an U1-trasphere 5 μm ODS reverse phase column and a UV detector at 254 nm. Recoveries for the 3 components are greater than 98%.

Simultaneous determination of diprophylline, proxyphylline and theophylline serum by reversed-phase high-performance liquid chromatography

A selective and reliable high-performance liquid chromatographic assay for the simultaneous determination of diprophylline, proxyphylline and theophylline is described. The method involves a single extraction procedure followed by separation on an ODS reversed-phase column using a ternary solvent system. The assay is sufficiently rapid and sensitive to be applied for pharmacokinetic studies as well as for routine monitoring of patient's serum after therapeutic doses of the combined preparation. The practicability and utility of the proposed method is demostrated in a pharmacokinetic study on four healthy volunteers.

Routine determination of hydroxyphenytoin in urine by high-performance liquid chromatography using an automatic column-switching technique

A high-performance liquid chromatographic method for the determination of the main phenytoin metabolite, hydroxyphenytoin, in the urine of epileptic patients is described. The use of an automated column-switching technique greatly simplifies the pretreatment steps. Thereby, both time and chemicals are saved. The possibility of error arising during the several pretreatment steps is considerably reduced. Following acid hydrolysis of the hydroxyphenytoin glucuronic acid conjugate the sample is diluted with water and after centrifugation is injected onto the pre-column. After washing for a short time with water, the substances which were absorbed on the head of the pre-column were backflushed with water—acetonitrile as eluent onto the analytical column. Separation is achieved by gradient elution using an ODS reversed-phase column with a particle size of 5 μm.

High Pressure Liquid Chromatographic Analyses of Sympathomimetic Bronchodilators in Inhalant Preparations

High pressure liquid chromatographic determination of clorprenaline hydrochloride, isoproterenol hydrochloride, orciprenaline sulfate, salbutamol sulfate and trimetoquinol hydrochloride in inhalant preparations was made using a Zorbax ODS reverse phase column and acetonitrile-phosphate buffer (pH 2.3) as mobile phase. The peaks of the additives and decomposition products in the solutions on chromatograms did not interfere with those of the bronchodilators. The coefficients of variation for analytical values, calculated from 2 different concentrations of the bronchodilators, ranged from 0.3 to 2.9%. Moreover, good recoveries (95.5-101.9%) were obtained from Alevaire or Bisolvon Solution.Thus, the methods can be applied to the evaluation of these inhalant preparations.

Pre-column derivatization with fluorescamine and high-performance liquid chromatographic analysis of drugs : Application to tocainide

The quantitative determination of tocainide, a new antiarrhythmic agent, by high-performance liquid chromatography (HPLC) is reported. The drug and a chemically similar internal standard were extracted from blood plasma with acetonitrile under salting-out conditions obtained by saturation of the aqueous medium with sodium chloride—sodium carbonate. The organic extract, without evaporation, was treated with borate buffer (pH 8.2) and fluorescamine. The resulting derivatives were chromatographed on an ODS reversed-phase column using a methanol—phosphate buffer (pH 7.0) mixture as mobile phase and were detected fluorometrically by monitoring the emission at 485 nm, with excitation at 395 nm. The intra-assay coefficients of variation were 3.0 and 4.3% for ten replicate 0.25 and 1.00 μg/ml samples, respectively, and the inter-assay coefficient of variation was 3.6% for ten replicate 1.00 μg/ml samples. The procedure is simple, rapid, sensitive, and specific. Several other drugs and drug metabolites also were derivatized with fluorescamine and chromatographed successfully. Pre-column derivatization with fluorescamine followed by HPLC with fluorometric detection may have significant advantages in drug analysis.

KININ FORMATION BY CATHEPTIC ENZYME IN RAT STOMACH

Four uterine-contractile substances (a, b, c and d) were extracted from procedures with acetic acid, n-butanol, distilled water, and methanol from the reaction mixture obtained by incubating rat plasma kininogen with the kininforming enzyme in the rat stomach. Gradient and equilibrium chromatography on SP-Sephadex C-25 columns were carried out with the extract containing these materials (a, b, c and d). The materials could not be separated by chromatography on SP-Sephadex C-25, but were separated by high performance liquid chromatography (HPLC) with a Zorbax ODS reversed-phase column. All the materials (a, b, c and d) contracted the rat uterus and relaxed the rat duodenum. The uterine-contractile activity of the materials was abolished by chymotrypsin, but not by trypsin treatment. The retention times of materials a, c and d on HPLC were different from that of kallidin, MLBK, bradykinin (BK) and neurotensin; but the retention time of material b was equal to that of BK. The content ratio of a: b: c: d was 4: 4: 67: 25, calculated from the uterine-contractile activity of these materials. The apparent molecular weight of the major material c, estimated by gel chromatography, was 1650. Material c contracted the rat uterus (1.2×10−10 g/ml), relaxed the rat duodenum (2×10−10 g/ml), and produced a fall in rabbit blood pressure (4.4×10−8 g/kg). Material c was classified as a biologically BK-like peptide which is distinct from kallidin, MLBK, BK and neurotensin.

Kinin formation by catheptic enzyme in rat stomach.

Four uterine-contractile substances (a, b, c and d) were extracted from procedures with acetic acid, n-butanol, distilled water, and methanol from the reaction mixture obtained by incubating rat plasma kininogen with the kininforming enzyme in the rat stomach. Gradient and equilibrium chromatography on SP-Sephadex C-25 columns were carried out with the extract containing these materials (a, b, c and d). The materials could not be separated by chromatography on SP-Sephadex C-25, but were separated by high performance liquid chromatography (HPLC) with a Zorbax ODS reversed-phase column. All the materials (a, b, c and d) contracted the rat uterus and relaxed the rat duodenum. The uterine-contractile activity of the materials was abolished by chymotrypsin, but not by trypsin treatment. The retention times of materials a, c and d on HPLC were different from that of kallidin, MLBK, bradykinin (BK) and neurotensin; but the retention time of material b was equal to that of BK. The content ratio of a: b: c: d was 4: 4: 67: 25, calculated from the uterine-contractile activity of these materials. The apparent molecular weight of the major material c, estimated by gel chromatography, was 1650. Material c contracted the rat uterus (1.2 x 10(-10) g/ml), relaxed the rat duodenum (2 x 10(-10) g/ml), and produced a fall in rabbit blood pressure (4.4 x 10(-8) g/kg). Material c was classified as a biologically BK-like peptide which is distinct from kallidin, MLBK, BK and neurotensin.

Chromatographic separation of benzo(a)pyrene isomers on nematic liquid crystal GC and polymeric reversed-phase HPLC phases

The use of two nematic liquid crystals (BABT and BPhBT) as GC stationary phases for the separation of monohydroxybenzo(a)pyrenes. as their TMS ethers, and monomethylbenzo(a)pyrenes was developed and compared with the separation of these isomers by HPLC using a polymeric ODS reversed-phase column. It was found that while HPLC and GC gave comparable separation of the hydroxy isomers, 10 out of 12 separated, better separation of the methyl isomers was obtained using HPLC. A simultaneous use of both HPLC and GC would resolve the twelve hydroxy isomers in about 70min. The results indicated that HPLC, using polymeric reversed-phase columns, is as powerful a tool as GC using nematic liquid crystal phases, for the separation of benzo(a)pyrene isomers. A discussion of the effect of solute length-to-breadth ratio on elution order is presented.

The determination of unconjugated estrone, estradiol, estriol and estetrol in serum or amniotic fluid by high performance liquid chromatography with an amperometric detector (author's transl)

A simultaneous microdetermination of unconjugated estrone, estradiol, estriol and estetrol in serum or amniotic fluid by High Performance Liquid Chromatography with an Amperometric Detector is described. Steroids in serum or amniotic fluid were extracted with 10 volumes of ethyl ether, and then ether extract was evaporated to dryness under N2 gas. After defatting with a mixture of 50% methanol/n-hexane, the methanol phase was evaporated to dryness under N2 gas. The residue was applied to microcolumn packed with 2 ml volume of Sephadex LH-20 in the eluting solvent benzene/methanol (85:15). Fractions contained estrone and estradiol; estriol and estetrol were collected and then evaporated to dryness under N2 gas. The sample solution was applied to HPLC using a reverse phase ODS column and acetonitrile: 0.1M KH2 PO4 47:53 for estrone and estradiol fraction, and 30:70 for estriol and estetrol fraction as a mobile phase, respectively. The fraction of each estrogen was separated completely within a 20 minute period. The limit of detection of estrone, estradiol, estriol and estetrol was 50 pg, respectively.

Determination of ethinylestradiol and norethisterone in an oral contraceptive capsule by reversed-phase high performance liquid chromatography.

A specific and sensitive analytical method is described for the simultaneous determination of ethinylestradiol and norethisterone in a capsule formulation. These steroids, commonly used in oral contraceptives, were extracted from the capsules with acetonitrile and tetrahydrofuran. The steroids were then quantitated with a high performance liquid chromatograph using a ODS reversed-phase column and a ternary solvent system of water, acetonitrile, and tetrahydrofuran as the mobile phase. Several solvent systems for the mobile phase were examined using various C18 columns. The k' values for several steroids are reported, together with column performance parameters. It was found that columns from different manufacturers had significantly different behaviors with respect to separation parameters for these steroids.